1. Field of the Invention.
The present invention relates to a process applied for the production of spherically shaped polymer particles (beads) on the basis of polyvinyl alcohol (PVAL) in which a magnetic colloid has been encapsulated that lends the polymer beads magnetic properties and enables these to bind biomolecules or cells.
2. Description of the Related Art.
In recent years, magnetic polymer beads have been used mainly in biochemistry and medicine to separate cells, proteins and nucleic acids. On account of their magnetic properties, they can also be used as transport systems for certain drugs in certain parts of the body. The use of magnetic beads has great practical advantages over conventional separation systems since the magnetic particles, which usually take the form of fine suspensions or emulsions, can be separated from the mixture by means of magnetic forces. This separation technique dispenses with normal centrifugation. The magnetic fractions can also be separated within one minute and are thus enormoursly time-saving compared to normal chromatographic column separation techniques. A vital part of the aforementioned technique is the time-consuming equilibration and elution processes which can practically be eliminated with the magnetic bead technique. A further important advantage of the magnetic bead technique is the manner of the reaction kinetics. Packing materials with particle sizes of 50-100 .mu.m are usually used in column chromatography. However, since the separation capacities are often inadequate for such particle sizes, there is an increasing tendency to use particle sizes of &lt;50 .mu.m and even &lt;10 .mu.m. In order to withstand the high pressures generated during the passage through the column, such media are practically no longer porous. This is the reason why a change had to be made in practice from the transparent plastic or glass columns to pressure-resistant steel columns. The powerful pump systems needed there are a further disadvantage of today's column chromatography technique. These drawbacks, which are eventually due to inadequate reaction kinetics, can be completely avoided through the use of the magnetic bead technology.
Through the use of finely dispersed PVAL particles with a particle size of 1-10 .mu.m, preferably 1-4 .mu.m, the particles remain in suspension for a number of hours so that the reaction kinetics correspond to those of a quasi-homogeneous solution. As a result of this stable suspension, stirring or shaking can also be dispensed with in most cases.
Processes to produce iron-dextrane microparticles are described in the U.S. Pat. No. 4,452,773. 30-40 nm large colloid iron oxide particles in which dextrane has been absorbed are obtained by mixing an Fe(II) and Fe(III) saline solution in the presence of a defined amount of dextrane and subsequently adding alkali. A similar process forms the basis of the PCT application WO 90/07380. Dextrane is added to Fe(II) and Fe(lII) saline solutions and treated at 40.degree. C. before being titrated with NaOH to produce superparamagnetic particles with a size of 40-100 nm. The disadvantage of both process is that separation is only possible by means of a high gradient magnetic field because of the fineness of the particles. This high gradient magnetic field is generated by a separating column densely packed with steel wool or similar microparticle substances which is placed between the pole shoes of two strong electromagnets or hand magnets. The particles are separated by passing the suspension through the packed separating column. A separation of such colloids is not possible with normal hand magnets. Therefore, there are in principle hardly any experimental differences between common chromatography techniques. A further disadvantage of the aforementioned process of production is that no uniform particle size can be obtained by the actual production process. This is only possible through a fractionated magnetic separation. Furthermore, detection of these magnetic particles is also complicated by the fact that the particles are no longer visible under a light-optical microscope. In a further process which forms the basis of U.S. Pat. No. 4,070,246, magnetic particles are obtained by converting p-aminobenzoic acid and an aldehyde through the addition of a ferromagnetic powder. The production of defined beads which are normally required for diagnostic tests, is not possible with this process. It is also not possible to chemically couple biomolecules to this carrier. The same applies to the process described in U.S. Pat. Nos. 4,106,448, 4,136,683 and 4,735,796 in which magnetic particles are encapsulated in dextrane for diagnostics and tumour therapy. The covalent coupling of biomolecules of the aforementioned process is also not described. U.S. Pat. No. 4,647,447 describes the production of ferromagnetic particles for NMR diagnostics. This process starts either with Fe(I)/Fe(III) saline solutions or directly with microparticle ferrites which are converted to magnetic suspensions in the presence of a complexing agent in the form of serum albumin, polysaccharides, dextran or dextrin. Other ferromagnetic particles which are encapsulated in a silane matrix are dealt with in U.S. Pat. No. 4,628,037. Superparamagnetic iron oxide, described in U.S. Pat. No. 4,827,945, is also used as contrast medium in NMR diagnostics. Coated magnetic particles can be produced with these substances through precipitation of Fe(II)/Fe(III) saline solutions by means of bases in the presence of serum albumin, polypetides or polysaccharides. The magnetic particles can be targeted into certain areas of the body by coupling specific antibodies to the matrix. The production of iron oxides through the precipitation of iron salts in the presence of dextranes or polyglutaraldehydes, for example, forms the basis of U.S. Pat. No. 4,267,234. All the aforementioned processes and products have one thing in common, namely, the ferromagnetic or superparamagnetic particles are only produced through the precipitation of a saline solution, which assumes a certain molecular ratio of Fe(II) and Fe(III) salts in the presence of a complexing or coating agent. The particles described display a rather wide range of particle sizes. Defined drop or spherical particles cannot be produced with the aforementioned processes. The materials described display an amorphous-like geometric structure. On account of their fineness, which is usually in the nm range, they are primarily suitable as a contrast medium for NMR diagnostics or as a cell marker. Moreover, the separation of the magnetic fractions is not usually possible with a simply hand magnet, such as is advantageous for fast diagnostic tests or affinity chromatography separations.
The preparation of magnetic albumin or protein microparticles coated with specific coupling agents which can be used for virus and cell separations as well as diagnostic tests, is described in U.S. Pat. Nos. 4,345,588; 4,169,804; 4,115,534; 4,230,685; 4,247,406 and 4,357,259.
Magnetic particles with a defined bead-shape structure are known from U.S. Pat. No. 4,861,705. The subject matter of the aforementioned patent are agarose polyaldehyde composite particles which are produced through a suspension of the polymer phase in an oil phase. Magnetic polymer particles with a particle size of 40-1000 .mu.m are obtained by admixing a ferrofluid, by definition a very fine superparamagnetic aqueous iron oxide colloid, to the polymer phase.
Perfectly bead-shaped particles are described in U.S. Pat. No. 4,654,267. The process differs fundamentally from the aforementioned in that polyacrylates or polystyrene, which is initially radically polymerised to bead-shaped particles by means of suspension polymerisation, is used as a matrix. The particles are then swelled in an organic phase under defined conditions. This is followed by an incubation of the polymer particles in an Fe(II)/Fe(III) saline solution that is oxidised to superparamagnetic iron oxide using ammonia once the particles have diffused. This process produces spherical particles with a particle size of between 0.5 and 20 .mu.m. The process itself is technically very complicated. Apart from the use of highly toxic substances, between 10 and 30 hours are required to prepare the basic matrix. Moreover, additional nitro, nitroso or amino groups are needed which are introduced into the polymer matrix in an additional preparation stage to guarantee an adequate absorption of the Fe salts. The great disadvantage of the particle described here is the basic polymer, polystyrene. Polystyrene is a very hydrophobic material with a strong tendency to unspecific absorption when in contact with protein solutions or other biomolecules. This phenomenon is disadvantageous particularly in immunoassays and affinity chromatography separations. The drawbacks of the aforementioned processes in terms of the production costs and effort, particle geometry, magnetic separation behaviour, properties of the polymer matrix or type of coupling process can be avoided by a novel water-in-oil suspension process. The polymer matrix used is polyvinyl alcohol (PVAL), which is suspended and cross-linked as an aqueous solution by stirring in an organic phase that cannot be mixed with water. Examples of such organic phases are generally known from the state-of-the-art in suspension polymerisation. Common vegetable oils are preferably used for the process in accordance with the invention. In order to achieve the desired magnetic properties of the polymer particles, the polymer phase is mixed with a magnetic colloid, e.g. iron oxide powders or ferrofluids, and then suspended in the oil phase. The production of bead-shaped PVAL particles through the suspension of an aqueous polymer solution is described in the Ger. Offen. 41 27 657. Magnetic particles can be produced by adding magnetite powder to the polymer solution. The aforementioned process uses polymer solutions and oil phases which contain no additives in the form of emulsifiers or other surfactants. Because of this, the particle sizes can quite easily be between 50 and 500 .mu.m. The particle sizes are primarily determined by the viscosity of the organic and/or polymer phase in the aforementioned process.